Antibodies have long been used in medical diagnosis, e.g., determining blood types, and in biological experimentation. The usefulness of antibodies, however, has been somewhat limited, as their complexity and diversity have made it very difficult to obtain homogeneous antibodies. Antibodies are complex protein or protein-based molecules which are produced by the immune systems of animals to protect the animal against foreign substances. Antibodies for medical use are generally obtained by injecting an animal with a foreign substance which will stimulate the animal's immune system, and, most commonly, isolating an antibody fraction from the peripheral blood serum or from the ascitic fluid. The antibody fraction contains antibodies specific to the injected foreign substance as well as various other antibodies produced by the animal, and by known techniques, it may be possible to substantially isolate an antibody specific to the particular foreign substance. However, even when an antibody for a particular foreign substance is isolated, such antibody is actually a mixture of several antibodies which recognize various antigenic determinants of the foreign substance or related substances. While some individual antibody molecules may be highly specific, recognizing only a certain foreign substance or portion thereof, other antibody molecules may be less selective, recognizing not only the subject foreign substance but other substances as well. Since it is generally practically impossible to separate all related antibodies, even the most carefully purified antibody fractions may react with more than one substance.
In recent years, techniques of producing monoclonal antibodies have been developed which make it possible to obtain homogenous, highly specific antibodies. Kohler G. and Milstein, C.: (1975) Nature 256 495-497. Generally, such antibodies are produced by immunizing an animal with a protein fraction or other foreign substance, obtaining antibody-producing cells from the animal, and fusing the antibody producing cells with strains of myeloma cells, e.g., tumor cells, to produce hybridomas which are isolated and cultured as monoclones. The monoclonal hybridomas may either be cultured in vitro or may be grown as tumors in a host animal. Since each antibody-producing cell produces a single unique antibody, the monoclonal cultures of hybridomas each produce a homogenous antibody which may be obtained either from the culture medium of hybridoma cultures grown in vitro or from the cells, ascitic fluid, or serum of a tumor bearing host animal.
Not all of the hybridoma clones which result from fusing neoplastic cells with antibody producing cells are specific for the desired foreign substance or antigen (a substance with which the antibody reacts) since many of the hybridomas will make antibodies which the animal has produced to react with other foreign substances. Even antibodies against the subject antigen will differ from clone to clone since antibodies produced by different cells may react with different antigenic determinants of the same molecule. From each clone, therefore, it is necessary to obtain the resulting antibody or the antibody-containing medium, serum or ascitic fluid and test its reactivity with the subject antigen and to test its specificity by determining with what other substances, if any, it recognizes. While the necessity of characterizing the antibody of each clone adds to the complexity of producing monoclonal antibodies, the wide variety of homogeneous antibodies which may be obtained gives investigators a number of very precise tools to map the structure and development of somatic cells.
The availability of homogeneous, highly specific monoclonal antibodies increases the value of antibodies as diagnostic, experimental and therapeutic tools. Use of monoclonal antibodies for tumor and virus detection have been described in U.S. Pat. Nos. 4,172,124 and 4,196,265.
Monoclonal antibodies are particularly suitable for studying the pathways and processes by which cells differentiate into different types of somatic cells to produce the various tissues of the body. Cell differentiation is a complex subject, and understanding of the processes are only beginning. Proteins which are specific to particular cell types and which may be detected by different monoclonal antibodies, serve as precise markers for the study of cell development and differentiation. Monoclonal antibodies which are specific for given proteins not only may be used to ascertain the presence of known proteins in a cell, but may be used to detect substances heretofore undiscovered. Theoretically it may be possible to eventually obtain monoclonal antibodies for every macromolecule in the body to permit the complete mapping of the various proteins, etc.
An important topic in the field of cell differentiation is the study of cells which, in their mature form, are non-proliferating being derived from actively proliferating stem cells. Many examples of such cells may be found in the peripheral blood. Red blood cells and leukocytes arise from stem cells in the bone marrow and both are normally non-proliferating as mature cells in the blood stream. Misdevelopment of somatic cells may lead to cancers, including blood cell related cancers such as myelomas and leukemias, and monoclonal antibodies are useful in determining the proteins present in such cell to more fully trace their development and derivation.
It is a primary object of the present invention to create and culture hybridomas which produce monoclonal antibodies that react with the human transferrin receptor blocking transferrin binding to the cells and interfering with the ability of the cells to proliferate. The long term objective is to use these antibodies to regulate cell growth in proliferative diseases in vivo particularly in the treatment of cancer.